Photosynthetic response and antioxidative activity of ‘Hass’ avocado cultivar treated with short-term low temperature

To investigate the effects of short-term low temperatures, three-year-old avocado (Persea americana cv. Hass) seedlings were treated with 1, − 2, or − 5 °C for 1 h and subsequently recovered in ambient condition for 24 h. Leaf color changes were investigated with chlorophyll, carotenoid, and phenolic contents. Photosynthetic responses were examined using gas exchange analysis. With H2O2 contents as oxidative stresses, enzymatic (ascorbate peroxidase, APX; glutathione reductase, GR; catalase, CAT; peroxidase, POD) and non-enzymatic antioxidant activities were determined using spectrophotometry. Leaves in the avocado seedlings started to be discolored with changes in the contents of chlorophyll a, carotenoids, and phenolics when treated with − 5 °C. However, the H2O2 content was not different in leaves treated with low temperatures. Photosynthetic activities decreased in leaves in the seedlings treated with − 5 °C. Of antioxidant enzymes, APX and GR have high activities in leaves in the seedlings treated with 1 and − 2 °C. In leaves in the seedlings treated with − 5 °C, the activities of all enzymes decreased. Non-enzymatic antioxidant activity was not different among leaves treated with low temperatures. These results indicated that APX and GR would play a critical role in withstanding chilling stress in ‘Hass’ avocado seedlings. However, under lethal temperature, even for a short time, the plants suffered irreversible damage with the breakdown of photosystem and antioxidant system.

for examining photosynthetic responses and antioxidant activities, compared before and after low-temperature treatments. Since the photosynthetic responses and antioxidant activities were measured using non-destructive and destructive methods, respectively, different avocado seedlings were used for each analysis. Before low-temperature treatment, six and three avocado seedlings were examined for photosynthetic responses and antioxidant activities, respectively, on February 20, 2021. Three biological replicates of each analysis were treated with low temperatures, including 1, − 2, or − 5 °C, for 1 h in dark chambers on March 15, 2021. Then all the plants were relocated to the greenhouse to recover from the chilling stress for 24 h before investigating photosynthetic responses and antioxidant activities. Photosynthetic responses were measured with a recently fully expanded leaf on each biological replicate. Antioxidant activities were biochemically assayed with five leaves of each replicate. These leaves were frozen in liquid nitrogen at stored at − 80 °C until use. Daily mean solar irradiance and temperature in the greenhouse were measured and recorded from January 01 to March 31, 2021 ( Fig. 1) using data loggers (WatchDog 2450, Spectrum Technologies, Inc., Aurora, IL, USA) with photosynthetically active radiation sensors (LightScout Quantum Light Sensor, Spectrum Technologies, Inc.).
Determination of total chlorophyll, carotenoid, and phenolic contents. Total chlorophyll and carotenoid contents were determined following the procedures of Lichtenthaler 25 . The absorbance of the extracts was measured at wavelengths of 661.1, 644.8, and 470 nm using a spectrophotometer (OPTIZEN™ Alpha, K Lab Co., Ltd., Daejeon, Republic of Korea). Chlorophyll and carotenoid contents were calculated based on µg g −1 fresh weight (FW) using Eqs. (1), (2), (3), and (4). www.nature.com/scientificreports/ Total phenolic contents were measured using Folin-Ciocalteu assay as described by George et al. 26 . To each extract concentration of leaf extracts (25,50,100,200, and 400 µg), 2.5 mL of Folin-Ciocalteu reagent (1/10 dilution) and 2 mL of 7.5% Na 2 CO 3 (w/v) solution were added and incubated at 45 °C for 15 min and the absorbance was read at a wavelength of 765 nm using a spectrophotometer (OPTIZEN™ Alpha, K Lab Co., Ltd., Daejon, Republic of Korea). The total phenolic content was expressed as mg gallic acid equivalents per gram of FW (mg GAE g −1 FW).
Gas exchange and chlorophyll fluorescence measurements. All gas exchange and chlorophyll fluorescence measurements were simultaneously conducted for recently fully expanded leaves using a portable photosynthesis system (LI-6400XT, Li-Cor, Co., Inc., Lincoln, NE, USA) equipped with a leaf chamber fluorometer (LI-6400-40), according to Rho, et al. 15 . CO 2 and light response curves (i.e., the net CO 2 assimilation (A)-the intercellular CO 2 concentration (C i ) and A-photosynthetic photon flux density (PPFD) curves) of the leaves were determined before and after the low-temperature treatments. During the measurements, the leaf chamber was maintained at 20 °C, and the relative humidity of the leaf chamber was at around 50%. We recorded A, transpiration rate (E), C i , stomatal conductance to H 2 O (g s ), the quantum yield of PSII estimated by fluorescence measurement (Φ PSII ), the quantum yield estimated by gas exchange measurement (Φ CO2 ), photochemical quenching (qP), and non-photochemical quenching (qN) over these measurements. The nomenclatures for chlorophyll fluorescence parameters were referred to Baker 27 . CO 2 response curves were constructed as a function of C i ranging from 0 to 1400 µmol CO 2 mol −1 . Under saturating light conditions of 1500 PPFD, the CO 2 concentration of the leaf chamber head was first set to 400 µmol CO 2 mol −1 (equivalent to ppm) and reduce to 200, 100, and 0 µmol CO 2 mol −1 to measure the photosynthetic responses at low CO 2 levels. Then, the C a was set back to 400 µmol CO 2 mol −1 and remained for 4 min to reacclimate RuBisCo to the ambient level of CO 2 . At 200 ppm intervals, the CO 2 level was increased to 600, 800, 1000, 1200, and 1400 µmol CO 2 mol −1 while the instrument's collecting the parameters. Light response curves were constructed as a function of incident PPFD ranging from 50 to 1500 µmol m −2 s −1 . Each PPFD was projected from internal red and blue light-emitting diodes with 10% blue light to maximize stomatal aperture and was maintained for 10 min for equilibration. The ambient CO 2 concentration of 400 µmol CO 2 mol −1 was used over the measurements. The light intensity of the leaf chamber was set to 0 µmol quanta m −2 s −1 PPFD to adapt the leaves in the dark for 20 min and then with the saturating pulse of 8000 µmol quanta m −2 s −1 PPFD for 0.5 s Fv/Fm was measured. The light intensity then was maintained at 1500 µmol quanta m −2 s −1 PPFD for 20 min to adapt the leaves in light to measure the light saturating photosynthesis and chlorophyll fluorescence values. The light intensity was decreased to 1200, 1000, 800, 600, 400, 200, 100, and 50 µmol quanta m −2 s −1 while the instrument's collecting the parameters.
Determination of H 2 O 2 contents. H 2 O 2 contents were determined as described by Shi et al. 28 . Leaves were homogenized in an ice bath with 0.1% trichloroacetic acid (TCA) using a tissue to TCA ratio at 1:10 (w/v). The homogenate was centrifuged at 12000×g for 15 min, and 0.5 mL supernatant was added to 0.5 mL of 10 mM potassium phosphate (pH 7.0) and 1 mL of 1 M KI. After 1 h in darkness, the absorbance was read at a wavelength of 390 nm, and the H 2 O 2 contents were calculated based on the standard curve.
Protein content was determined with bovine serum albumin as a standard, according to Bradfor 30 . Protein content and enzyme activities were measured by using a spectrophotometer (OPTIZEN™ Alpha).
APX activity was measured as described by Nakano and Asada 31 using a reaction mixture containing 50 mM potassium phosphate (pH 7.0), 0.2 mM H 2 O 2 , and 0.5 mM ascorbate. The H 2 O 2 -dependent oxidation of ascorbate was monitored following the absorbance decrease at a wavelength of 290 nm. The enzyme activity was calculated by using the extinction coefficient of 2.8 mM −1 cm −1 .
CAT activity was measured according to Mishra et al. 32 . A reaction mixture composed of 50 mM potassium phosphate (pH 7.0) and 11 mM H 2 O 2 . The decomposition of H 2 O 2 was measured at a wavelength of 240 nm, and the enzyme activity was calculated by using the extinction coefficient of 0.036 mM −1 cm −1 .
POD activity was determined by measuring the absorbance increase at a wavelength of 470 nm due to the formation of tetraguaiacol in a reaction mixture containing 40 mM potassium phosphate (pH 7.0), 1. www.nature.com/scientificreports/ guaiacol, and 6.5 mM H 2 O 2 as described by Chance and Maehly 33 . The enzyme activity was calculated by using the extinction coefficient of 26.6 mM −1 cm −1 . GR activity was measured by oxidized GSH-dependent oxidation of NADPH. The reaction mixture contained 100 mM potassium phosphate (pH 7.8), 2 mM EDTA, 0.2 mM NADPH, and 0.5 mM GSSG 34 . The absorbance change at a wavelength of 340 nm was monitored and the enzyme activity was calculated by using the extinction coefficient of 6.22 mM −1 cm −1 .
Determination of non-enzymatic antioxidant activity. Non-enzymatic antioxidant activity was determined using ABTS radical scavenging assay, according to Re et al. 35 . ABTS was dissolved in distilled water to a 7 mM concentration. ABTS radical cation was produced by reacting the ABTS stock solution with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 12 h. The stock solution (25 µL) was diluted with 70% methanol (22 mL). The sample (50 µL) was mixed with the ABTS solution (1 mL) for 3 min. The absorbance was read at a wavelength of 734 nm. A c is the absorbance of the control, reacted with ethanol (50 µL) and the ABTS solution (1 mL). A s is the absorbance of the sample. The ABTS radical scavenging capacity was calculated using Eq. (5).
Statistical analysis. All statistical analyses were performed using R Statistical Software version 4.1.3 36 and RStudio IDE version 2022.02.1 + 461 37 with agricolae packages version 1.3-5 38 . Statistically significant differences were determined by analysis of variance. Means were compared using the Tukey's honestly significant difference test at p < 0.05. Linear correlations between selected variables were determined by Pearson correlation coefficient (r), after Shapiro-Wilks test for validating the normal distribution of the variables. The difference was considered significant at p < 0.05.

Results and discussion
Visual appearances and pigment changes. 'Hass' avocado seedlings were visually changed before and after treatment with 1, -2, or -5 °C (Fig. 2). The color of leaves in the seedlings treated with 1 and -2 °C maintained green as same as leaves before treatment, while those treated with -5 °C turned yellow or brown. Since avocado is an evergreen fruit tree, the color changes would be a visual signal for physiological disorders and leaf senescence 39 . The discoloration of leaves in the seedlings treated with -5 °C for 1 h was shown in various avocado cultivars, including 'Geada' , 'Fortuna' , 'Fuerte' , 'Quintal' , 'Margarida' and 'Primavera' 40 . In grafted oneyear 'Hass' avocado, exposure from -2 to -3 °C for 10 h led to the discoloration and death of leaves within one to three days 3 . It can also accompany the degradation of pigment, protein, and cell wall components, and a decrease in photosynthesis 39 . Although the visual damage showed a part of plant status since morphological changes would not fully describe the degree of damage, biochemical and physiological investigation are required to identify tree conditions exposed to various low temperatures.
The pigment compositions of leaves changed under low temperatures in various plants 20 . In the present study, we determined the changes in the concentrations of chlorophyll a and b, carotenoids, and phenolics ( Table 1). The contents of chlorophyll a in the leaves in the seedlings treated with 1 and -2 °C did not change compared to the leaves before treatment, while those treated with -5 °C significantly decreased. The contents of chlorophyll b and total chlorophyll did not change between those before and after treatments (Table 1). Compared to leaves before treatment, the ratio of chlorophyll a to b decreased up to about 50% in the leaves in the seedlings treated with -5 °C, but those treated with 1 and -2 °C were not significantly different. Chlorophyll a dominates in photosystems, while chlorophyll b content is restricted to the light harvesting complex 20 . Therefore, a decrease in the chlorophyll a in the leaves in the seedlings treated with -5 °C indicated that at a specific low temperature, even for a short time, whole photosystems were damaged rather than specific sites such as the light harvesting complex.
When plants were exposed to low temperatures, secondary metabolites were synthesized to protect plant cells from ROS, which can cause changes in leaf color 20 . Total carotenoids and phenolics in the leaves in the seedlings treated with -5 °C were accumulated more than the others (Table 1). Carotenoids and phenolics have been associated with the photoprotection against chlorophyll oxidation by ROS in the photosystems and light harvesting complexes 20 . Especially, phenolic compounds have dual roles as antioxidants and as substrates for oxidative browning reactions with oxidoreductases, including polyphenol oxidase (PPO) and POD 41 . They are functional as antioxidants at relatively low concentrations while, at higher concentrations, since they themselves are susceptible to oxidation, they can behave as pro-oxidants due to their involvement in initiation reactions. The main oxidative phenomenon (usually deteriorative) of this type is enzymatic browning which involves initial enzymatic oxidation of phenolic compounds located predominantly in the vacuole by PPO located in cytoplasm to form slightly colored quinones. Although the oxidation of phenolic compounds leads to yellowing or browning, the products are considered antioxidants. Therefore, the accumulation of carotenoids and phenolics in the leaves in the seedlings treated with -5 °C were associated with severe damages compared to those treated with 1 and -2 °C.
Oxidative stress. H 2 O 2 contents, the index of oxidative stress, increased in the leaves in avocado seedlings treated with low temperatures (Fig. 3). The H 2 O 2 contents in all the leaves were about four-fold higher than before treatment; however, the contents were not significantly different among the leaves in avocado seedlings treated with low temperatures. Enhanced ROS generation is a well-known effect of cold stress by low temperature 42 . www.nature.com/scientificreports/ When the ambient temperature becomes lower, the light becomes excessive due to the reduction of the photosynthetic activities, so there is excessive production of excited chlorophyll, which generates ROS. However, the effect of increased ROS generation on plant physiology varies from plant species, the level and duration of low temperatures, and other growing conditions. In this study, only avocado seedlings treated with -5 °C did not survive (Fig. 1), despite the same levels of H 2 O 2 contents among leaves in avocado seedlings treated with different low temperatures; the H 2 O 2 contents were also not consistent with pigment contents (chlorophyll a, r = -0.74 non-significant (ns) ; chlorophyll b, r = -0.47 ns ; total chlorophylls, r = -0.88 ns ; total carotenoids, r = 0.53 ns ; total phenolics, r = 0.67 ns .). These results indicated that avocado seedlings would have different physiological or biochemical responses against oxidative stress at each low temperature.
Leaf gas exchange properties. Before short-term low temperature treatments, the 'Hass' avocado leaves showed signature responses of CO 2 response curves (i.e., A-C i curves; A, net CO 2 assimilation rate as a function of C i , intercellular CO 2 concentration of leaves) in C 3 plants having both RuBisCo-limited photosynthesis up to approximately from 0-500 µmol CO 2 mol −1 air and RuBP regeneration-limited photosynthesis from 500 µmol CO 2 mol −1 air and above (Fig. 4) 43 . Triose-phosphate utilization-limited photosynthesis, however, was not observed in the samples, which may or may not be observed in C 3 plants 44 . The maximum level of A was around 5 μmol CO 2 m −2 s −1 observed at 1,100 µmol CO 2 mol −1 air. Avocado trees have relatively low photosynthesis compared with other C 3 fruit trees, showing various ranges of maximum photosynthetic rates under different growing conditions 45 . The actual A values under the ambient growing conditions at 400 µmol CO 2 m −2 s −1 with saturating light intensity of 1500 PPFD were lower than those reported in the literature, measured 1.6 µmol CO 2 m −2 s −1 before treatment (Figs. 4 and 5; Table 2). The A values of container-grown avocado were reported to be around 6 µmol CO 2 m −2 s −1 in three-month-old avocado seedlings 46 and 5.9-7.6 µmol CO 2 m −2 s −1 in less than one-year-old grafted avocado seedlings 3 . As avocado plants grow A also increases, so it could reach up to Table 1. Changes in chlorophyll, carotenoid, and phenolic contents of three-year-old 'Hass' avocado seedling leaves before treatment and 24 h after treated with 1, − 2, and − 5 °C for 1 h. 1 Means with standard errors from three replicates with five leaf each. 2 Means within columns followed by different lowercase letters are significantly different according to Tukey's honestly significant difference test at p < 0.05. 3 Statistical significance codes: *, **, and *** for p < 0.05, 0.001, and 0.001 levels, significantly. After short-term low temperature treatments, the avocado leaves showed distinguished down-regulation of photosynthetic activity in the seedlings treated with − 5 °C while those in the seedlings treated with the other low temperatures did not inhibit the photosynthetic responses. At low levels of C i , less than 400 µmol CO 2 mol −1 air, net CO 2 assimilation turned to negative values, meaning that the leaves rather were respiring over the low C i range. When subtropical or tropical plants were exposed to low temperatures, 0-12 °C can cause chilling injuries, while < 0 °C can cause more acute frost injuries, represented by the inhibition of photosynthesis from the ROS production 3 . In 'Hass' avocado, it was shown that an overnight chilling temperature reduced A about 65% on a subsequent day after the cold night 3 .
The avocado seedlings showed a characteristic of shade leaves, A being saturated at 500 µmol m −2 s −1 photosynthetic photon flux density (PPFD) as reported in Mandemaker 45 (Fig. 5). The maximum level of A under saturating light intensity was around 2 µmol m −2 s −1 before treatment, which is lower than the previously reported values 3 . After short-term low temperature treatments, as shown in the A−C i responses (Fig. 4), the light response  www.nature.com/scientificreports/ curves (i.e., A-PPFD curve) presented an apparent down-regulated photosynthetic capacity in the seedlings treated with − 5 °C under saturating light conditions. Over all the PPFD measured, A remained negative, indicating the leaves were damaged after the − 5 °C treatment were respiring at all light levels. However, similarly to the A-C i responses, the 1 and − 2 °C treatments both did not produce a significant reduction in A, although these are damaging low temperatures in avocado demonstrated in the literature 3 . This is likely because the plants used in our study were adapted to low temperatures for a few months even before treatment (average growing temperature of 10.53 °C and 11.06 °C for the two months prior to the experiment). Under the ambient growing conditions at 400 µmol CO 2 m −2 s −1 with saturating light intensity of 1500 PPFD, the avocado leaves in the seedlings treated with − 5 °C showed lower gas exchange and chlorophyll fluorescence values in A, E, g s , Φ PSII , Φ CO2 , and Fv/Fm and higher values in C i , qP, and qN in comparison with the leaves in the seedlings before treatment and treated with 1 and − 2 °C ( Table 2). The changes in these values indicate not only reduced photosynthetic CO 2 assimilation and transpirational H 2 O release but also increased photoprotection operating to compensate for the decreased utilization of absorbed light energy harvested in PSII by chilling stress from low temperature. A notable decrease in g s of the leaves in the seedlings treated with − 5 °C confirms the stomatal limitation of photosynthetic CO 2 uptake. The subsequent negative A value along with increased C i in the leaves in the seedlings treated with − 5 °C implies the damaged tissues by chilling stress only afforded respiration, not photosynthesis. Also, the non-stomatal limitation was observed in the following manner. Increased qP in the leaves in the seedlings treated with − 5 °C could be likely from increased photochemical quenching of absorbed light energy through photorespiration, not through photosynthesis. Such down-regulation, in turn, appeared to induce non-photochemical quenching of the excess energy, increased qN in the leaves in the seedlings treated with − 5 °C. These photosynthetic alterations were reported in the literature as adaptive responses to low temperature 47,48 .
Overall, from the response curve analysis, it was observed that the exposure to short-term low temperature led to stomatal and non-stomatal limitation of photosynthesis in avocado leaves, which only occurred at the leaves in the seedlings treated with − 5 °C, not at 1 and − 2 °C. In response to the downregulation of photosynthesis, the photoprotection in the leaves in the seedlings treated with − 5 °C was induced; nevertheless, the damaged PSII could not withstand saturating light intensity.

Antioxidant activities.
In 'Hass' avocado seedlings, enzymatic antioxidant activities were assayed to examine whether individual antioxidant enzymes contribute to the stabilization of photosystems against oxidative stress by short-term low temperatures (Fig. 6). Since the activity of individual enzymes varied depending on plant types and growth conditions 17,40,42 , the activity would be too complicated to understand. Still, the relationship with photosystem in this study could be described as relatively more straightforward than other biochemical systems. Generally, an increase in the activities prevented the accumulation of H 2 O 2 and consequent cellular damage, suggesting that enzymes may play an important role in plant responses to oxidative stress caused by low temperature 40 . In the leaves in the avocado seedlings treated with 1 and − 2 °C, APX and GR activities increased compared to those before treatment (Fig. 6A,B), while CAT and POD activities did not differ (Fig. 6C,D). This result suggests that ascorbate-glutathione pathway 49 using APX and GR was stimulated to protect photosystem from oxidative stress in the leaves in the seedlings treated with 1 and − 2 °C. Especially, APX is known to be allocated to chloroplasts, cytosol, and even in peroxisomes 50 . Chloroplast APX existing as both soluble and thylakoid-bound forms provided effective elimination of H 2 O 2 generated both internally and externally 50 . Although Table 2. Descriptive and inferential statistics of photosynthetic parameters at the operational points of the CO 2 (Fig. 2) and the light (Fig. 3) response curves. The means of biological replications are provided with the standard errors of the means in parentheses (n = 6). F-statistics of one-way ANOVA test results for lowtemperature effect (df = 2) on these photosynthetic parameters are presented with statistical significance codes. The corresponding p-values of the F-statistics are presented in parentheses. 1 A, the net CO 2 assimilation rate; E, transpiration rate; C i , the intercellular CO 2 concentration; g s , stomatal conductance to H 2 O; Φ PSII , the quantum yield of photosystem II estimated by fluorescence measurement; Φ CO2 , the quantum yield of CO 2 assimilation estimated by gas exchange measurement; Fv/Fm, the maximum quantum efficiency of photosystem II reaction centers; qP, photochemical quenching; qN, non-photochemical quenching. 2 Means with ± 1 standard errors of the means from six replicates-three from A-C i and A-PPFD curves each under the ambient 400 ppm CO 2 and saturating light 1500 PPFD conditions. 3 Means within columns followed by different lowercase letters are significantly different according to Tukey's honestly significant difference test at p < 0.05. 4 Statistical significance codes: *, **, and *** for p < 0.05, 0.001, and 0.001 levels, significantly. www.nature.com/scientificreports/ CAT and POD had antioxidant activity in response to chilling stress in other avocado cultivars 40 , the enzymes in 'Hass' avocado seedlings were not differently activated (Fig. 6C,D). A decrease in the enzyme activities occur when enzymes were consumed to counteract the increased in ROS production in response to stress or were inactivated due to severe cell damage 40 . In the leaves in avocado seedlings treated with − 5 °C, the activities of APX, GR, and CAT decreased compared to those before treatment and treated with 1 and − 2 °C (Fig. 6A-C). POD activities significantly decreased compared to those not before treatment but treated with 1 and − 2 °C (Fig. 6D). A decrease in the enzymatic activities had been shown in plants, including avocados, when it was exposed to close to the lethal temperature 40 . Nevertheless, we need further study to identify the sequence of decreases in the activities of antioxidant enzymes in the leaves in the seedlings treated with − 5 °C.
In addition to the stimulation of enzymatic antioxidants, non-enzymatic antioxidants could be accumulated to scavenge ROS generated by chilling stress (Fig. 7) 19 . Compared to avocado seedling leaves before treatment, non-enzymatic antioxidant activity did not change in those treated with 1 and − 5 °C but increased significantly in those treated with − 2 °C. Although the contents of carotenoid and phenolics were the highest in those treated www.nature.com/scientificreports/ with − 5 °C than the others (Table 1), the antioxidant activity could not be affected by the accumulation of the compounds. Weil et al. 51 reported, in 'Hass' avocado cultivar, a similar result with this study; the non-enzymatic antioxidant activity did not change under gradient decreased temperatures from 13 to − 2.9 °C with the duration of 2 h per each step. Meanwhile, the antioxidant activity of 'Ettinger' avocado cultivar, the greater cold tolerance than that of 'Hass' , was greater than before treatment when exposed to the gradient temperature. In various plants, cold-tolerant cultivars have higher or an increase in antioxidant activity under low temperatures than cold-sensitive cultivars 51 . In this result, we confirmed 'Hass' avocado characteristics as a cold-sensitive cultivar; the non-enzymatic antioxidant activity was not stimulated regardless of the level of chilling stress, even under critical temperature, which caused the severe damage of photosystem.

Conclusions
We investigated the effects of short-term low temperatures on physiological and biochemical responses in 'Hass' avocado cultivar for the cultivation in Republic of Korea, one of the new cultivation areas in temperate regions, where avocado has been just introduced. After being treated with 1 or − 2 °C for 1 h, the photosynthetic characteristics of the avocado leaves did not change compared to the leaves before treatment. Although oxidative stress occurred, the photosystem was protected by antioxidant enzymes, including APX and GR. After being treated with − 5 °C for 1 h, we identified the changes in morphology and pigment contents as a signal of physiological disorders. The avocado leaves showed remarkable reductions in photosynthetic activities; stomatal and nonstomatal limitations on photosynthesis lowered the capacity to process the light quanta. Under low-temperature conditions at − 5 °C, the excess light energy damaged PSII of the leaves with inactivated antioxidative mechanisms. These results indicated avocado seedlings suffer irreversible damage when exposed to the threshold level of low temperature, even for a short period of time. Therefore, 'Hass' avocado seedlings would be cultivated at least above − 5 °C, being a lethal temperature in the new cultivation regions of Republic of Korea.

Data availability
All data generated or analyzed during this study are included in this published article in form of figures and tables. . ABTS scavenging activity of three-year-old 'Hass' avocado seedling leaves before and after 24-h after exposure to 1, − 2, and − 5 °C for 1 h. Means with standard errors from three replicates with five leaf each. Means with bars followed by different lowercase letters are significantly different according to Tukey's honestly significant difference test at p < 0.05.